This invention relates to the field of nucleic acid and protein detection and, more specifically, to the rapid and accurate identification of organisms by detecting differences in nuclear and organellar introns.
Microorganisms are the cause of damaging infections in both plants and animals. About 1.3% of patients admitted to hospitals in the U.S. have positive fungal cultures. In particular, Candida albicans is one of the most frequently observed pathogens in immunocompromised patients. Most individuals are colonized with C. albicans as a commensal organism, and when the individual becomes immunocompromised, the organism can establish an infection. Systemic Candida infections extend hospital stays and contribute to increased mortality.
There is a need for epidemiological and diagnostic tools to detect infectious microorganisms in situations where they are hard to distinguish or where the nature of the agent is still under investigation. This is particularly true in fungal diseases where considerable effort has gone into studying and combating such diseases in immunocompromised human patients and in diseases of crops.
Epidemiological and diagnostic tools for classifying plant infecting and mammalian infecting fungi have been used to identify the origin of fungal infections and to track the progression of disease after treatment with antifungal drugs. In the case of mammalian fungal pathogens, there are at least 19 species of Aspergillus and at least seven species of Candida that cause infection. Almost all the xe2x80x9cspeciesxe2x80x9d in these genera are defined solely by morphological and nutritional characteristics. These tests are laborious and expensive and have not provided sufficient discrimination to date to classify all infectious organisms.
A variety of detection and identification methods have more recently been developed for detecting Candida albicans, including the germ tube test, carbohydrate assimilation test, antigen test, serology, fluorescein-conjugated lectin visualization, and nucleic acid detection by polymerase chain reaction (PCR). Despite these tests, current diagnosis of Candida continues to rely on differential culturing, because non-culture tests are costly, requiring multiple enzymatic or hybridization steps and, in the case of PCR, a series of different reaction cocktails and conditions. This additional work diminishes the throughput of a clinical laboratory and increases the chance of error.
There are no less than 30 genera of fungi involved in plant diseases and the relationships among these various species and genera of fungi is still not fully understood. Almost all the xe2x80x9cspeciesxe2x80x9d in plant fungal genera are presently defined by morphological features or by host range. However, the lack of good morphological characters in fungi has led to often opposing classifications based on host plants, as for in xe2x80x9cforma specialis,xe2x80x9d or other characters for sub-species groupings. Furthermore, in some cases, fungal morphological features can only be discerned when infections are well established on the plant host and symptoms are visible, or when the fungi are present in large enough quantities to be cultured from the plant. Thus, diagnostics of plant infecting fungi is at a rudimentary stage and early detection in asymptomatic plants is not possible using these methods.
Molecular-based methods have been applied to a very limited number of plant pathogenic fungi (reviewed by Swaminathan et al., in Diagnostic Molecular Microbiology, Principles and Applications, D H Persing et al. eds., ASM Press, Washington, D.C., pp 26-50 (1993)). For example, immunoassays have been devised for earlier detection of Pythium (Miller et al., Phytopathol. 78:1516 (1988)), Phytophthora and Rhizoctonia (MacDonald et al., Plant Disease 74:655-659 (1990)) and Mycosphaerella fijiensis (Novartis, AG Crop Protection Division, Basal Switzerland). Also, commercial kits are available and certified testing laboratories provide enzyme-linked immunoadsorbent assay (ELISA)-based assays for detection of some fungal species.
Furthermore, a variety of nucleic acid protocols have been used to detect plant pathogens, including plasmid content, pulsed field gel electrophoresis, nucleic acid hybridization, restriction digestion, and PCR (reviewed in Maclean et al., Adv. Plant Path., 10:207-244 (1993); van Belkum et al., Clin. Infect. Dis., 18:1017-1019 (1994); and Tang et al., Clin. Chem., 43:2021-2038 (1997)). A few examples of the application of these approaches to fungal pathogens in plants include the arbitrarily primed PCR (xe2x80x9cAPPCRxe2x80x9d or random amplified polymorphic DNA: xe2x80x9cRAPDxe2x80x9d)xe2x80x94based identification for epidemiology and population studies of intersterility groups in Heterobasidion annosum (Garbelotto et al., Can. J. Bot., 71:565-569 (1993)) and RAPD-based identification of pathogenic versus non-pathogenic isolates of Fusarium oxysporum formal specialis (f. sp.) dianthi (Manulis et al., Phytopath., 84:98-101 (1994)).
In addition, probes developed from tandem repeat loci within satellite DNA have been used to detect polymorphisms among Heterobasidion annosum isolates (DeScenzo et al., Phytopath., 84:534-540 (1994)).
Although each of these methods are useful, there currently is no single effective approach for detection and classification. Moreover, many of the methods require some foreknowledge of the particular species of organism likely to be present. It is apparent that a need exists for improved molecular methods that avoid the increased costs and reduced speed associated with present diagnostic and epidemiological tests for determining infection of plants and animals.
Accordingly, it is an object of the present invention to provide an approach to identify nucleic acid sequences and associated proteins that are useful for readily characterizing target organisms, such as differentiating between taxonomic groupings of target organisms, identifying the taxonomic group to which an organism belongs, etc. It also is an object of the present invention to use such nucleic acid sequences to rapidly and effectively identify organisms that are present in a sample. It is another object of the present invention to provide isolated nucleic acids comprising intronic regions useful in the methods of the invention. It is yet another object to provide kits suitable for practicing the methods of the invention.
To accomplish these and other objectives, there has been provided, according to one aspect of the present invention, a method for characterizing nuclear and organellar intronic regions that differ between or among various taxonomic groupings of organisms.
In one embodiment, an intronic region is selected from aligned nucleotide sequences of one or more gene homologs.
In another embodiment, a primer pair is generated for amplifying the intronic region and an amplified product is generated in a primer extension reaction. The amplified product from intronic regions of known organisms are analyzed to determine if the intronic region will be useful for characterizing unknown organisms. In one embodiment, the intronic region-specific primers flank more than one intron insertion site while in another embodiment, the intron region-specific primers flank a single intron insertion site.
In yet another embodiment, the intronic region is selected from gene sequences of organisms that reflect a broader taxonomic grouping than the taxonomic grouping of the target organisms sought to be characterized.
In still yet another embodiment, the target organisms sought to be characterized are from a single genus or very related genera and the organisms from which gene sequences are obtained are from different taxonomic classes or subclasses of organisms.
In further embodiments, the analysis of the amplified products from primer extension reactions include determining the presence or absence of the intronic region, the length of the intronic region, the nucleotide sequence of the intronic region, or restriction fragment length polymorphism. In some of these embodiments, the amplified product is detected by hybridizing with specific nucleic acid probes.
In yet a further embodiment, the nucleotide sequence of an intronic region identified from above is used to prepare intronic region-specific primers that are complementary to a sequence of nucleotides in the DNA of a particular target organism.
In an additional embodiment, intronic regions that contain an open reading frame encoding a protein (intronic region encoded protein: xe2x80x9cIREPxe2x80x9d) are detected by generating specific antibodies to the protein or by detecting enzymatic activity of the protein.
The present invention also provides methods to detect the presence of a particular organism in a sample based on characterizing its intronic region sequences. In accordance with this aspect of the present invention, intronic region sequences are detected by nucleic acid detection approaches including primer extension, probe hybridization and other methods. In primer extension reactions, the intronic region-specific primers flank more than one intron insertion site while in another embodiment, the intron region-specific primers flank a single intron insertion site.
In other embodiments, the analysis of the amplified products from primer extension reactions include determining the presence or absence of the intronic region, the length of the intronic region, the nucleotide sequence of the intronic region, or restriction fragment length polymorphism. In some of these embodiments, the amplified product is detected by hybridizing with specific nucleic acid probes.
In yet another embodiment, intronic region-specific primers that are complementary to a sequence of nucleotides in the DNA of a particular target organism are used in primer extension at high stringency.
In accordance with another aspect of the present invention, an intronic region comprising all or a portion of an open reading frame is detected by detecting the encoded protein (IREP) using antibodies specific for the encoded protein or by detecting enzymatic activity characteristic of the protein.
The present invention also provides isolated nucleic acids, comprising an intronic region from a fungal gene, which can be used as a probe and to express the encoded protein.
The present invention also provides the amino acid sequences of fungal mitochondrial intronic region open reading frames that can be used to raise anti-IREP antibodies of the invention and can be expressed to determine an associated enzymatic activity.
The present invention further provides kits for practicing the methods of the invention.
Other objects, features and advantages of the present invention will become apparent from the following detailed description.